Primitive and proximal hepatic stem cells

ABSTRACT

Hepatic progenitors comprise two populations of human hepatic stem cells, primitive and proximal hepatic stem cells, and two populations of committed progenitors, one for biliary cells and one for hepatocytes. Human primitive hepatic stem cells are a very small fraction of the liver cell population and give rise to proximal hepatic stem cells constituting a much larger fraction of the liver. Human proximal hepatic stem cells give rise to biliary and hepatocyte committed progenitors. Primitive and proximal stem cells are the primary stem cells for the human liver. Human primitive hepatic stem cells may be isolated by immunoselection from human livers or culturing human liver cells under conditions which select for a human primitive hepatic stem cell. Proximal hepatic stem cells may be isolated by immunoselection, or by culturing human liver cells under conditions which include a developmental factor. Proximal hepatic stem cells may also be isolated by culturing colonies comprising a primitive hepatic stem cell under conditions which include a developmental factor. Resulting compositions may be used for treating liver disorders and for producing bioartificial organs.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.11/609,729, filed Dec. 12, 2006, which is a Divisional of U.S.application Ser. No. 10/387,547, filed Mar. 14, 2003, now U.S. Pat. No.7,413,897, which claims priority from Provisional Application U.S.Application 60/365,361, filed Mar. 15, 2002. All application of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to human hepatic stem cells, pluripotentcells that give rise to mature liver cells. These include two stem cellpopulations: a very primitive progenitor, ductal plate stem cells, thatgive rise to proximal hepatic stem cells, the proximal stem cells thatgive rise to hepatocytes and biliary cells. The present invention alsorelates to methods of isolating the human hepatic ductal plate stemcells and to isolating proximal hepatic stem cells and committedhepatocytic progentitors and committed biliary progenitors. Compositionscomprising cells of the present invention can be used for cell and genetherapies and for the establishment of bioartificial organs.

BACKGROUND OF THE INVENTION 1. Anatomy of the Human Liver

The primary structural and functional unit of the mature liver is theacinus, which in cross section is organized like a wheel around twodistinct vascular beds: 3-7 sets of portal triads (each with a portalvenule, hepatic arteriole, and a bile duct) for the periphery, and withthe central vein at the hub. The liver cells are organized as cellplates lined on both sides by fenestrated endothelia, defining a seriesof sinusoids that are contiguous with the portal and centralvasculature. Recent data have indicated that the Canals of Hering, smallducts located around each of the portal triads, produce tiny ductulesthat extend and splice into the liver plates throughout zone 1 forming apattern similar to that of a bottle brush (Theise, N. 1999 Hepatology.30:1425-1433).

A narrow space, the Space of Disse, separates the endothelia fromhepatocytes all along the sinusoid. As a result of this organization,hepatocytes have two basal domains, each of which faces a sinusoid, andan apical domain which is defined by the region of contact betweenadjacent hepatocytes. The basal domains contact the blood, and areinvolved in the absorption and secretion of plasma components, while theapical domains form bile canaliculi, specialized in the secretion ofbile salts, and are associated through an interconnecting network withbile ducts. Blood flows from the portal venules and hepatic arteriolesthrough the sinusoids to the terminal hepatic venules and the centralvein.

Based on this microcirculatory pattern, the acinus is divided into threezones: zone 1, the periportal region; zone 2, the midacinar region, andzone 3, the pericentral region. Proliferative potential, morphologicalcriteria, ploidy, and most liver-specific genes are correlated withzonal location (Gebhardt, R., et al. 1988. FEBS Lett. 241:89-93;Gumucio, J. J. 1989, Vol. 19. Springer International, Madrid; Traber, P.et al. 1988. Gastroenterology. 95:1130-43). Gradients in theconcentration of blood components, including oxygen, across the acinus,and following the direction of blood flow from the portal triads to thecentral vein, are responsible for some of this zonation, for example thereciprocal compartmentation of glycolysis and gluconeogenesis. However,the periportal zonation of the gap junction protein connexin 26 and thepericentral zonation of glutamine synthetase, to name only two, areinsensitive to such gradients, are more representative of mosttissue-specific genes and appear to be determined by factors intrinsicto the cells or to variables other than blood flow in themicroenvironment.

In addition to hepatocytes, bile duct epithelial cells (cholangiocytes),and endothelial cells, the region between the portal and central tractscontains other cell types, such as Ito cells and Kupffer cells. Theseplay prominent roles in pathogenic conditions of the liver, especiallyin inflammation and fibrosis, but their direct contribution to the mainhomeostatic functions of the normal organ are apparently small.

2. Development of the Human Liver

The liver develops as a result of the convergence of a diverticulumformed from the caudal foregut and the septum transversum, part of thesplanchnic mesenchyme. The formation of the hepatic cells begins afterthe endodermal epithelium interacts with the cardiogenic mesoderm,probably via fibroblast growth factors. The specified hepatic cells thenproliferate and penetrate into the mesenchyme of the septum transversumwith a cord like fashion, forming the liver anlage. The directepithelial-mesenchymal interaction is critical in these earlydevelopmental stages of the liver and dictates which cells will becomehepatocytes or cholangiocytes, and the fenestrated endothelia,respectively. Mutations in the mesenchyme-specific genes hlx and jumonjiblock liver development, illustrating the importance of contributionsfrom this tissue. Early in its development, the liver consists ofclusters of proximal hepatic stem cells bounded by a continuousendothelium lacking a basement membrane and abundant hemopoietic cells.As the endothelium is transformed to become a discontinuous, fenestratedendothelium, the vasculature, especially the portal vasculature, becomesmore developed with the production of basement membranes. The portalinterstitium may provide the trigger for the development of bile ducts,and as it surrounds the portal venules, hepatic arterioles, and bileducts, portal triads are formed. Proximal hepatic stem cells rapidlyproliferate and parenchymal plates are formed, probably in response tochanges in the amount and distribution of such tissue-organizingmolecules as C-CAM 105, Agp110, E-cadherin, and connexins, coincidentwith the relocation of most, but not all, of the hemopoietic cells tothe bone marrow. Recent studies suggest that some hemopoieticprogenitors persist in the adult quiescent rodent liver, and hemopoieticstem cells have been isolated from both adult human and murine liver(Crosbie, O. M. et al. 1999. Hepatology. 29:1193-8).

The rat liver forms in embryonic life at about day 10, referred to as“embryonic day 10” or E10, with the invagination of the cardiacmesenchyme by endoderm located in the midgut region of the embryo(Zaret, K. 1998. Current Opinion in Genetics & Development. 8:526-31).Earliest recognition of liver cells in the embryos has been achieved byusing in situ hybridization studies for mRNA encoding alpha-fetoprotein(AFP) ((Zaret, K. 1998. Current Opinion in Genetics & Development.8:526-31; Zaret, K. 1999 Developmental Biology (Orlando). 209:1-10).AFP-expressing cells are observed in the midgut region of the embryonear the mesenchyme that produces the heart on day 9-10 in all rat andmouse livers assayed. The liver becomes macroscopically visible by E12and is about 1 mm in diameter by E13.

In parallel, hemopoiesis occurs with the first identifiable hemopoieticcells appearing by E15-E16 (in rodents) and by the 3^(rd) to 4^(th)month (in humans) and with the peak of erythropoiesis (formation oferythroid cells or red blood cells) occurring by E18 (in rodents) and bythe 5^(th)-6^(th) month (in humans). At the peak of erythropoiesis, thenumbers of these red blood cells dominate the liver and account for morethan 70% of the numbers of cells in the liver. The end of thegestational period is on day 21 in rodents and 9 months in humans.Within hours of birth, the numbers of hemopoietic cells declinedramatically such that by 2 days postnatal life (rodent) and within aweek or two (human), the vast majority of the hemopoietic cells havedisappeared having migrated to the bone marrow. No one knows the causefor the migration of the hemopoietic cells. There are however twodominant speculations.

First, the hemopoietic progenitors prefer relatively anaerobicconditions and most of them migrate to the bone marrow (which isrelatively anaerobic) with the elevated oxygen levels in the liver withthe activation of the lungs. In addition, there have speculations thatthe loss of the pregnancy hormones may also be a factor in themigration. Postnatally, the loss of the hemopoietic progenitors in theliver is correlated with a dramatic reduction in the numbers of hepaticprogenitors and a parallel increase in the numbers and maturity of thehepatocytes and biliary cells. Full maturity of the liver is completedby 2-3 weeks postnatal life (in rodents) and within a few months(humans). By then the remaining hepatic progenitor cells are localizedto the regions of Canals of Hering, with the dominant numbers of thempresent the portal triads in the periphery of each liver acinus (Thieseet al, Crawford et al.).

Thereafter, the classic architecture of the liver acinus is establishedwith each acinus being defined peripherally by six sets of portaltriads, each one having a bile duct, an hepatic artery and an hepaticvein, and in the center a central vein that connects to the vena cava.Plates of liver cells, like spokes in a wheel, extend from the peripheryto the center. By convention, the plates are divided into three zones:Zone 1 is near the portal triads; zone 2 is midacinar; and zone 3 isnear the central veins. The only diploid cells of the liver are in zone1; tetraploid cells are in zone 2; and tetraploid, octaploid andmultinucleated cells are in zone 3. The pattern is highly suggestive ofa maturational lineage that ends in an apoptotic process (Sigal, S. H.,S. et al. 1995. Differentiation. 59:35-42).

3. Liver Disease

Each year in the United States, there are about 250,000 peoplehospitalized for liver failure. Liver transplants are curative for someforms of liver failure, and approximately 4100 transplants are performeda year in United States. One of the limiting factors in livertransplantation is the availability of donor livers especially given theconstraint that donor livers for organ transplantation must originatefrom patients having undergone brain death but not heart arrest. Liversfrom cadaveric donors have not been successful, although recent effortsto use such donors have supported the possibility of using them if theliver is obtained within an hour of death.

Cell transplantation into the liver is an attractive alternative therapyfor most liver diseases. The surgical procedures for celltransplantation are minor relative to those needed for whole organtransplantation and, therefore, can be used for patients with varioussurgical risks such as age or infirmity. The use of human liver cells issuperior to liver cells derived from other mammals because the potentialpathogens, if any, are of human origin and could be better tolerated bypatients and could be easily screened before use.

Attempts to perform liver cell transplantation have made use ofunfractionated mature liver cells and have shown some measure ofefficacy (Fox, I. J. et al. 1998. New England Journal of Medicine.338:1422-1426). However, the successes require injection of largenumbers of cells (2×10¹⁰), since the cells do not grow in vivo.Furthermore, the introduction of substantial numbers of large matureliver cells (average cell diameter 30-50 μm) is complicated by theirtendency to form large aggregates upon injection, resulting inpotentially fatal emboli. Moreover, these cells elicit a markedimmunological rejection response forcing patients to be maintained onimmunosuppressive drugs for the remainder of their lives. Finally,mature liver cells have not been successfully cryopreserved andcomplicated logistics are required to coordinate the availability ofsuitable liver tissue, the preparation of cell suspensions and theimmediate delivery of the cells for clinical therapies.

4. Totipotent Stem Cells

Stem cells are an alternative cell-based therapy for liver disease.Totipotent stem cells are primitive cells that can self-replicate, arepluripotent, i.e. produce daughter cells with more than one fate, thatcan expand extensively and that can give rise to determined stem cellsthat can reconstitute a tissue or tissues. Most of the literature onstem cells derives either from the literature on embryos or that onhemopoietic, epidermal, or intestinal tissues.

More recently, the definitions have been modified to recognizeparticular classes of stem cells. Those with the potential toparticipate in the development of all cell types including germ cellsare referred to as totipotent stem cells and include the zygote andnormal embryonic cells up to the 8 cell stage (the morula). Embryonicstem cells, also called “ES” cells, consist of permanent cellpopulations derived from totipotent, normal cells in blastocysts, thatwere first reported in the early 1980s. ES cell lines can be cultured invitro with maintenance of totipotency. When ES cells are injected backinto normal blastocysts, they are able to resume embryonic developmentand participate in the formation of a normal, but chimeric, mouse.Although ES cell lines have been established from many species (mouse,rat, pig, etc.), only the mouse system has been used routinely togenerate animals with novel phenotypes (knockouts, transgenics) bymerging modified ES cells from culture to blastocysts and thenimplanting the blastocysts into pseudopregnant hosts. Embryonic germ(EG) cell lines, which show many of the characteristics of ES cells, canbe isolated directly in vitro from the primordial germ cell population.As with ES cells, the EG cells contributed to chimeras, including thegerm line, when injected into blastocysts.

Recent, highly publicized experiments have reported that human ES cellcultures can be established from human embryos. It has been suggestedthat these human ES cells may be injected into tissues in the hope thatthey will be able to reconstitute damaged organs and tissues. However,ES and EG cells are tumorigenic if introduced into immunocompromisedhosts in any site other than in utero, forming teratocarcinomas.Therefore, the plan to inoculate human ES cells into patients isunrealistic and with the grave possibility of creating tumors in thepatients. To overcome this impasse, some groups are pursuing the plan ofdifferentiating the ES cells under defined microenvironmental conditionsto become determined stem cells that can then be safely inoculated intopatients. For example, there is some measure of success in generatinghemopoietic progenitors. However, the concern remains that residual EScells in the culture could pose the risk of tumorigenesis, if thecultures are inoculated into a patient. In summary, until research indevelopmental biology reveals the myriad controls dictating the fates ofcells during embryogenesis, the ES cells will remain as an experimentaltool with little hope for clinical programs in cell or gene therapies.The only realistic option for clinical programs in cell and genetherapies is to use determined stem cells in which the genetic potentialis restricted to a limited number of cell types.

5. Determined Stem Cells

Determined stem cells are pluripotent cells that have restricted theirgenetic potential to that for a limited number of cell types and haveextensive growth potential. Increasing evidence such as that from thetelomerase field suggest that determined stem cells do not, strictlyspeaking, self-replicate, that is their progeny can have less growthpotential than the parent. Determined stem cells give rise to committedprogenitors, daughter cells that lose pluripotency by restricting theirgenetic potential to a single fate, e.g. hepatocytes, whose committedprogenitors are referred to as committed hepatocytic progenitors. In thehepatic lineage there are committed hepatocytic progenitors (giving riseto hepatocytes) and committed biliary progenitors (giving rise to bileducts).

The transitions from the stem cell to the adult cells occur in astep-wise process yielding a maturational lineage in which cell size,morphology, growth potential and gene expression is tied to the lineage.The metaphor of aging is useful in defining the process. The “young”cells have early gene expression and the greatest growth potential; thecells late in the lineage have “late” gene expression and usually arelimited in their growth or do not grow at all. The late cells can beconsidered “old” or in biological terms, apoptotic, and ultimately aresloughed off. The maturational lineage process results in a naturalturnover for the tissue and allows for regeneration after injuries.Tissues differ in the kinetics of the maturational process. Thematurational lineage of the gut is quite rapid with a complete cycleoccurring in less than a week; that of the liver is slow occurring, andin the rat liver is about a year.

There is a strong clinical and commercial interest in isolating andidentifying immature progenitor cells from liver because of the impactthat such cell population may have in treating liver diseases. The useof hepatic progenitors in cell and gene therapies can overcome many ofthe shortcomings associated with use of mature liver cells describedabove. The cells are small (7-15 μm), therefore minimizing the formationof large emboli. Also, the cells have extensive growth potential meaningthat fewer cells are needed for reconstitution of liver tissue in apatient. Finally, the progenitors have minimal antigenic markers thatmight elicit immunological rejection providing hope that little or noimmunosuppressive drugs might be needed.

6. Isolation of Liver Progenitors

Isolation of liver progenitors from liver is known to be an extremelychallenging task due to the shortage of markers that positively selectfor liver cells. The only available antibodies for candidates of hepaticprogenitors are those monoclonal antibodies that are prepared againstsubpopulations of hepatic progenitors, called oval cells if isolatedfrom hosts exposed to oncogenic insults. These antibodies howevercross-react with antigens present in hemopoietic cells.

The term oval cells is derived from a myriad of studies in the fields ofcarcinogenesis and oncogenesis. Animals exposed to carcinogens or otheroncogenic insults experience a dramatic loss of mature liver cells(killed by the various insults) and, secondarily, expansion of smallcells (7-15 μm in diameter) with oval-shaped nuclei and bearing markersthat comprised both hepatic and hemopoietic antigens (Grisham andThorgeirrson, 1998). The studies on oval cells led to the hypothesesthat they are hepatic progenitors that are triggered to expand under theconditions of the oncogenic insults and that with the proper conditionscan go on to be tumor cells. The phenotype of the oval cells varies insubtle and not subtle ways depending on the oncogenic insult(s).Moreover, they are known to be readily established in culture withoutspecial feeders or medium conditions. (J. Grisham and S. Thorgeirrson,1998, Hepatic Stem Cells, In: Stem Cells, C Potten, editor, AcademicPress, NY). Based on these findings and on studies characterizing someof the cell lines derived from the oncogenic treatments, it was realizedthat liver tumors are malignantly transformed progenitors and that ovalcells are partially or completely transformed progenitors (Zvibel I,Fiorino A, Brill S, and Reid LM. Phenotypic characteriztaion of rathepatoma cell lines and lineage-specific regulation of gene expressionby differentiation agents. Differentiation 63:215-223, 1999).

Attempts have been made in the past to obtain the hepatic progenitorcell population, suggested to be the most versatile population for celland gene therapy of the liver. U.S. Pat. Nos. 5,576,207 and 5,789,246(Reid et al.) utilize cell surface markers and side scatter flowcytometry to provide a defined subpopulation in the liver.Subpopulations of rat hepatic cells have been isolated by removal oflineage-committed cells followed by selection for immature hepaticprecursors which were detected as being agranular cells bearingOC.3-positive (oval cell antigenic marker), AFP-positive,albumin-positive, and CK19-negative (cytokeratin 19) cell markers. Theforegoing rat liver subpopulations demonstrate particularcharacteristics important in isolation and identification of enrichedhepatic progenitors from rodent liver.

Thus, there exists a need to develop methods of isolating human hepaticprogenitors that may be used to treat patients with liver disease ordysfunction. The present invention satisfies this need and providesmethods of treatment as well.

SUMMARY OF THE INVENTION

The present invention is directed to a composition comprising a humanprimitive hepatic stem cell that is a precursor to a proximal hepaticstem cell, hepatocytic progenitor, or biliary progenitor. The humanprimitive hepatic stem cell of the invention expresses ep-CAM, AC133,and albumin.

Another embodiment of the present invention is a composition comprisinga human proximal hepatic stem cell that is a precursor to a hepatocyticor biliary progenitor. The human proximal hepatic stem cell of theinvention expresses alpha-fetoprotein, albumin, and cytokeratin 19.

Another embodiment of the present invention is a method for isolating ahuman hepatic progenitor comprising identifying a cell that expressesep-CAM and AC133. The human hepatic progenitor isolated by the presentmethod preferably expresses albumin. In a preferred embodiment of thepresent invention, the isolated human hepatic progenitor is a stem cell,preferably a primitive hepatic stem cell or a proximal hepatic stemcell.

Another embodiment of the present invention is a method for isolating ahuman primitive hepatic stem cell comprising culturing a mixture ofcells derived from human liver tissue on a surface under conditionswhich select for a hepatic stem cell with serum-free media comprising aregulator of carbohydrate metabolism, an iron carrier, and a membraneproducing factor, whereby a colony is formed comprising a humanprimitive hepatic stem cell. In a preferred embodiment of the presentinvention, the isolated human primitive hepatic stem cell expressesep-CAM, AC133, and albumin, and preferably further expresses cytokeratin8/18 and cytokeratin 19.

Another embodiment of the present invention is a method for isolating ahuman proximal hepatic stem cell comprising culturing a mixture of cellsderived from human liver tissue on a surface under conditions whichselect for a hepatic stem cell with serum-free media comprising aregulator of carbohydrate metabolism, an iron carrier, and a membraneproducing factor, whereby a colony is formed comprising a humanprimitive hepatic stem cell, and culturing the cells from the colonywith a developmental factor. In a preferred embodiment of the presentinvention, the isolated human proximal hepatic stem cell expressesalpha-fetoprotein, albumin, and cytokeratin 19. In a preferredembodiment of the present invention, the developmental factor isprovided by a secondary cell, preferably a feeder cell, preferably anSTO feeder cell, an endothelial cell, or a stromal cell.

Another embodiment of the present invention is a method for isolating ahuman proximal hepatic stem cell comprising culturing a mixture of cellsderived from human liver tissue under conditions which select for ahepatic stem cell with serum-free media comprising a regulator ofcarbohydrate metabolism, an iron carrier, and a membrane producingfactor, whereby a colony is formed comprising a human primitive hepaticstem cell, and culturing the cells from the colony with a developmentalfactor. In a preferred embodiment of the present invention, the isolatedhuman proximal hepatic stem cell expresses alpha-fetoprotein, albumin,and cytokeratin 19. In a preferred embodiment of the present invention,the developmental factor is provided by a secondary cell, preferably afeeder cell, preferably an STO feeder cell, an endothelial cell, or astromal cell.

Another embodiment of the present invention is an isolated primitivehepatic stem cell. Yet another embodiment of the present invention is anisolated human proximal hepatic stem cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates colony formation on plastic culture from enrichedfetal parenchymal cells on plastic culture Day 1 (Top Panel) and Day 5(Bottom Panel).

FIG. 2 demonstrates colony formation on plastic culture from Day 5 toDay 14.

FIG. 3 demonstrates colony behavior on plastic culture.

FIG. 4 demonstrates staining of colony cells on plastic for albumin (row1), CK19 (row 2), ep-CAM (row 3) and NCAM (row 4).

FIG. 5 demonstrates staining for colonies cultured on plastic for CD146and CD133 (top), and AC133 (bottom).

FIG. 6 demonstrates a primary culture of proximal stem cells after 7days on a STO feeder layer staining for albumin (row 1),alpha-fetoprotein (row 2), and CK19 (row 3).

FIG. 7 demonstrates the development of a colony cell removed fromplastic culture and plated onto a STO cell feeder layer.

FIGS. 8-11 demonstrate eruption of cells from the colony on an STOfeeder layer.

FIGS. 12 and 13 demonstrate the enrichment of AFP-expressing cells inhuman liver cells.

FIGS. 14-18 demonstrate the isolation of a subpopulation of adult humanliver cells co-expressing albumin, CD133, and Ep-CAM.

FIG. 19 depicts the growth curve of 9 stem cell colonies from 3 liverscultured on plastic over a 3 week period. Growth measurements startedafter 12 days in culture. The curve shows that the cells grow with adoubling time of 5.2 days.

FIG. 20 depicts Western blots of albumin (ALB, upper group) and alphafetoprotein (AFP, lower group) expression in freshly isolated fetalliver cells and during their subsequent culture on plastic substratum.

In the left hand grouping two cell fractions are shown (P and I) basedupon centrifugation through Ficoll. Cells which pelleted in the Ficollare designated P, and cells that become layered at the interface betweenaqueous medium and Ficoll are designated I. The single middle blot showsthe albumin and AFP expression in purified colony cells (primitivehepatic stem cells) cultured on plastic for 3 weeks. The right handpanel shows control lanes in which there was either no protein (blank),albumin (ALB) or alpha fetoprotein (AFP) standards. 10 ug of protein wasloaded in each lane.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

In the description that follows, a number of terms are used extensivelyto describe the invention. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms, the following definitions are provided.

CD: “Cluster of differentiation” or “common determinant” as used hereinrefers to cell surface molecules recognized by monoclonal antibodies.Expression of some CDs are specific for cells of a particular lineage ormaturational pathway, and the expression of others varies according tothe state of activation, position, or differentiation of the same cells.

Cell Therapy: As used herein, the term “cell therapy” refers to the invivo or ex vivo transfer of defined cell populations used as anautologous or allogenic material and transplanted to, or in the vicinityof, a specific target cells of a patient. Cells may be transplanted inany suitable media, carrier or diluents, or any type of drug deliverysystems including, microcarriers, beads, microsomes, microspheres,vesicles and so on. They can also be used in a bioreactor in which theyprovide critical functions and the bioreactor used as an assist devicefor patients with liver dysfunction(s).

Committed Progenitors: Highly proliferative cells that that gives riseto daughter cells of only one fate. A “biliary committed progenitor”gives rise to bile ducts and can be recognized antigenically by theexpression of cytokeratin 19, but not AFB. A “hepatocytic committedprogenitor” gives rise to hepatocytes and can be recognizedantigenically by the expression AFP and albumin, but not cytokeratin 19.The commitment process is not understood on a molecular level. Rather,it is recognized to have occurred only empirically when the fates ofcells have narrowed from that of a predecessor.

Gene Therapy: As used herein, the term “gene therapy” refers to the invivo or ex vivo transfer of defined genetic material to specific targetcells of a patient, thereby altering the genotype and, in mostsituations, altering the phenotype of those target cells for theultimate purpose of preventing or altering a particular disease state.This can include modifying the target cell ex vivo and introducing thecells into the patient. Alternatively, a vector can be targeted to liverprogenitor cells in vivo to deliver the exogenous genetic material andtransfect the progenitors. Furthermore, genetically engineeredprogenitor cells can be used in a bioreactor as a therapy for patientsor as source of biological products. As this definition states, theunderlying premise is that these therapeutic genetic procedures aredesigned to ultimately prevent, treat, or alter an overt or covertpathological condition. In most situations, the ultimate therapeuticgoal of gene therapy procedures is to alter the phenotype of specifictarget cell population.

Hepatic Cells: A subpopulation of liver cells which includes hepatocytesand biliary cells.

Hepatic Progenitors: A subpopulation of stem cells, these cellsultimately give rise to mature parenchymal cells that comprisehepatocytes and biliary cells. The hepatic progenitors include thefollowing two subpopulations: (a) hepatic stem cells and (b) committedprogenitors.

Hepatic Stem Cells: A subpopulation of hepatic progenitors, including“primitive hepatic stem cells” and “proximal hepatic stem cells”.

Precursor: As used herein, the term “precursor” refers to a first typeof cell that gives rise to a second type of cell. The precursor maydirectly give rise to the second type of cell. The precursor may alsogive rise to the second type of cell, through one or more otherintermediary cell types.

Primitive Hepatic Stem Cells: As used herein, the term “primitivehepatic stem cells” refers to hepatic stem cells that give rise toproximal hepatic stem cells.

Proximal Hepatic Stem Cells: As used herein, the term “proximal hepaticstem cells” refers to hepatic stem cells that give rise to hepatocytesand biliary epithelial cells.

Liver Cells: As used herein, the term “liver cells” refers to all typeof cells present in normal liver, regardless of their origin or fate.

Stem Cells: As used herein, the term “stem cells” refers to highlyproliferative cells that can give rise to daughter cells with more thanone fate, that is they are pluripotent. Totipotent stem cells, such asembryonic stem cells (ES cells) or embryonic cells up to the 8 cellstage of a mammalian embryo, have self-renewal (self-maintaining)capacity in which the stem cell produces a daughter cell identical toitself. By contrast, determined stem cells, such as hemopoietic,neuronal, skin or hepatic stem cells, are pluripotent and have extensivegrowth capacity but have questionable self-renewal capacity. In the caseof totipotent stem cells, some daughter cells are identical to theparent, and some “commit” to specific fate(s) restricting their geneticpotential to that which is less than the parent's. In the case ofdetermined stem cells, some daughter cells retain pluripotency and somelose it, committing to a single, specific fate.

When the terms “one,” “a,” or “an” are used in this disclosure, theymean “at least one” or “one or more,” unless otherwise indicated.

2. Diagnostic Markers for Hepatic Lineages

Alpha-fetoprotein (AFP) and albumin, both cytoplasmic proteins, areespecially reliable markers for hepatic lineages when assayed asproteins. Messenger RNAs encoding variant forms of these proteins areexpressed in hemopoietic progenitors but are not translated; forexample, a variant form of AFP mRNA differing from that in hepatic cellsby replacement of the exon 1 encoded sequences with either an alternateexon 1 or two exons (Kubota, Storm and Reid, submitted; also in patentapplication). Therefore, the expression of these two proteins is thefoundation for identification of the hepatic subpopulations from othercell types in the liver. Within the developing liver the presence of AFPand albumin is recognized as a strong positive indicator of hepaticprogenitor cells. In the earliest stages of liver development thesecells are capable of producing offspring that enter both biliary andhepatocyte lineages. If these daughter cells commit to the biliarylineage AFP expression ceases. However, AFP expression persists in thehepatocyte lineage until the perinatal period when it is suppressed,leaving albumin expression as one of the principal characteristics ofthe adult hepatocyte.

3. Processing of Human Liver Progenitors

The isolation of liver cells usually involves enzymatic and mechanicaldissociation of the tissue into single cell suspensions followed byfractionation with density gradient centrifugation, centrifugalelutriation, differential enzymatic digestion protocols (i.e., hepaticstellate cells), and/or with selection using cell culture (reviewed inFreshney, “Culture of Animal Cells, A Manual of Basic Technique” 1983,Alan R Liss, Inc. NY). Liver tissue may be obtained from a fetus, aneonate, an infant (birth to 1 year old), a child (1 year old topuberty), or an adult (beyond puberty). Density gradient centrifugationis preferably used to fractionate and isolate different cell populations(e.g., hepatoblasts).

4. Culturing of Proximal Hepatic Stem Cells and Other Progenitors

Proximal hepatic stem cells and committed hepatic progenitors requireembryonic liver stromal feeders and a serum-free medium supplementedwith a mixture of defined hormones and growth factors [1-6]. Clonogenicexpansion and prolonged maintenance of key markers of the proximalhepatic stem cells, of committed progenitors and of diploid adult livercells can occur if the embryonic liver stromal feeders are substitutedwith STO feeder cells in combination with a serum-free, hormonallydefined medium supplemented with insulin, transferrin/Fe and preferablyhydrocortisone [7]. Given that these conditions support a diverse rangeof progenitors from fetal tissue and even colony formation of diploidadult cells [7], different conditions are needed to select for theprimitive hepatic stem cells

5. Isolation of Primitive Hepatic Stem Cells

The present invention involves a method of isolating primitive hepaticstem cells from human liver tissue comprising applying a cell suspensionderived from liver tissue, preferably enriched for parenchymal cells, toa plastic surface and subjecting the cells to stringent cultureconditions that eliminate mature liver cells, the proximal hepatic stemcells and the committed progenitors. Stringent culture conditionsinclude the use of serum-free medium supplemented with a regulator ofcarbohydrate metabolism, a source of iron, a membrane producing factor,and preferably an anti-oxidant.

A preferred regulator of carbohydrate metabolism is insulin. A preferredsource of iron is transferrin. A preferred membrane producing factor isa composition comprising one or more lipids, most preferably, free fattyacid. A preferred anti-oxidant is selenium. The serum-free medium ispreferably further supplemented with hydrocortisone. The liver tissue ispreferably obtained from a fetus, a neonate, an infant, a child, ajuvenile, or an adult, and most preferably, from a fetus.

Primitive hepatic stem cells are isolated by culturing the liver-derivedcell suspension on a plastic surface at low cell densities (e.g.1000-2000 cells/cm²). The stringent culture conditions result inemergence of primitive hepatic stem cells from human liver which areprecursors to proximal hepatic stem cells. These primitive hepatic stemcells from human liver co-express Ep-CAM, AC133, CK8/18, CK19, andalbumin and subpopulations of them express N-CAM, CAM 5.2, and c-kit.

One of skill in the art will recognize that the present invention may beused to isolate primitive cells from other tissue types.

6. Isolation of the Proximal Hepatic Stem Cells

Human proximal hepatic stem cells give rise to hepatocytes or biliaryepithelia, or combinations thereof. Human proximal hepatic stem cellsco-express Ep-CAM, CK8/18, cytokeratin 19, alpha-fetoprotein, andalbumin and subpopulations express AC133. Human proximal hepatic stemcells can be isolated by various methods, including (i) immunoselectionof cells that co-express EP-CAM (ii) culturing liver derived cellsuspensions, preferably enriched for parenchymal cells, with adevelopmental inducing factor, or (iii) culturing human primitivehepatic stem cells with a developmental inducing factor. Thedevelopmental inducing factor is preferrably provided by a secondarycell. Preferred secondary cells include an STO feeder cell, an embryonicliver stromal cell, or an endothelial cell.

7. Isolation of Hepatic Progenitor by Immunoselection

The present invention also involves a method of isolating a hepaticprogenitor from liver-derived cell suspensions based on immunoselectingcell surface markers specific for a hepatic progenitor. A hepaticprogenitor may be isolated according to the present invention byselecting for cells that express ep-CAM, and preferably those cells thatfurther express AC133. The immunoselected hepatic progenitor of thepresent invention preferably further express albumin, and morepreferably further express cytokeratin 19. Preferably, theimmunoselected hepatic progenitor is a stem cell.

In one embodiment of the present invention, the isolated hepaticprogenitor is a primitive hepatic stem cell. In another embodiment ofthe present invention, the isolated hepatic progenitor is a proximalhepatic stem cell.

8. Production of Hepatic Progenitors

The present invention also involves a method of producing proximalhepatic stem cells and committed progenitors from hepatic primitivehepatic stem cells comprising either directly plating onto STO feederlayers and in the HDM or by transferring the primitive hepatic stemcells from colonies on culture plastic to a STO feeder layer andallowing the proximal hepatic stem cells to emerge from the colonies ofprimitive hepatic stem cells.

Proximal hepatic stem cells and committed progenitors may also beproduced from primitive hepatic stem cells by culturing on uncoatedsurfaces, including petri dishes (preferably non-charged polystyrenesurfaces), tissue culture plastic (preferably polystyrene surfacesexposed to ionizing gas so that the polystyrene is polarized with apreferential orientation of negative (or positive) charges towards theside to which the cells are to attach), microcarriers (preferablyculture beads to which cells can be bound), textile fabrics (preferablynylon, cotton, polyester), synthetic scaffoldings (preferably made frompolylactides, poly (propylene fumarate), poly(ortho esters), or othersynthetic materials) or sponges (preferably natural or syntheticsponges).

Proximal hepatic stem cells and committed progenitors may also beproduced from primitive hepatic stem cells by culturing on biologicalsurfaces. The biological surfaces can be coated or prepared onto thesurfaces in the categories above. Thus, for example, one can coatextracellular matrix coatings onto petri dishes, tissue culture plastic,microcarriers or textile fabrics. Biological surfaces used in thepresent invention include (i) extracellular matrix (a complex mixture ofproteins and carbohydrates produced by cells and located outside andbetween cells and comprising collagens, adhesion proteins,proteoglycans, and other proteins), (ii) extracellular matrix components(individual, purified matrix components used alone or in combinationsfor optimization of cell attachment, growth and/or expression oftissue-specific function(s), including fibronectin, laminin, collagens(there are more than 20 families of collagens) including type Icollagen, type III collagen, type IV collagen (these three are the mostcommonly used today in cell culture), cell adhesion molecules or “CAMs”some of which are calcium-dependent and some of which are not, andproteoglycans (molecules that consist of a core protein to which isattached one or more glycosaminoglycan chains, polymers of a dimericunit of glucuronic acid or iduronic acid+an aminosugar). These includechondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, heparansulfate proteoglycan, heparin proteoglycan), (iii) tissue extractsenriched in extracellular matrix, inlcuding Matrigel (a urea extract ofa transplantable murine embryonal carcinoma. Can be coated onto any ofthe surfaces given in group I)), ECM (extraction of cultured cells usingdilute alkali, dilute detergent, high salt extraction, urea, etc. andleaving behind an exudate enriched in extracellular matrix componentscoating the surface (any of those in group I)), amniotic membrane matrix(extraction of amnions with dilute alkali, dilute detergent, high saltextraction, urea, etc. and leaving behind matrix components present inthe amnions), and biomatrix (extraction of tissue with high salt(e.g. >3 M NaCl) and nucleases to leave behind all the tissue'scollagens and any associated components such as adhesion proteins), (iv)serum coating (if one coats petri dishes or tissue culture dishes withserum, one adds adhesion proteins, especially fibronectin, present inhigh levels in serum), and (v) polylysine or polyleucine (coating withthese positively charged amino acids is used to attach epithelial cellspreferentially).

Proximal hepatic stem cells and committed progenitors may also beproduced from primitive hepatic stem cells by culturing under conditiondescribed in Anthony Atala and Robert P. Lanza, editors. Methods ofTissue Engineering. Academic Press, New York 2002, which is incorporatedherein byreference. Hepatic progenitors produced by the presentinvention include primitive and proximal hepatic stem cells, hepatocyticcommitted progenitors, and biliary committed progenitors.

9. Therapeutic Approaches

The isolated progenitors of the present invention may be used forliver-directed cell and/or gene therapy or as cells to be hosts forvirus production (e.g. hepatitis C) to generate vaccines. Also, theprogenitors of the present invention may be expanded ex vivo from liverbiopsies (e.g. punch biopsy) and the expanded cells used for autologousor allogeneic cell or gene therapies or used to seed bioreactors tocreate bioartificial livers that can be used clinically or for academicstudies. This would eliminate the necessity for major invasive surgicalresection of the patient's liver.

Once the progenitors are established in culture, gene transfer may beperformed using any of a number of different gene delivery vectorsystems. The growing characteristics of the progenitors of thisinvention permits the use in an ex vivo gene transfer using certain genedelivery vectors (i.e., retroviral vectors) which will require cellproliferation for efficient gene insertion and expression.

An alternative approach for gene therapy is to design vectors thattarget the progenitors specifically and then to inject the vector,coupled with the gene of interest, directly into the patient. Thevectors would target and modify the endogenous progenitor cellpopulation.

The progenitor of this invention can be used in an autologous orallogeneic liver-directed cell or gene therapy. Clearly, the use ofautologous hepatic progenitors will eliminate a significant concernregarding rejection of the transplanted cells. The progenitors of thisinvention are particularly attractive for allogenic cell transfer,because their antigenic profile suggests minimal immunological rejectionphenomena.

Once the autologous or allogenic progenitors are isolated purified andcultured, they can be genetically modified or remain intact, expanded invitro, and then transplanted back into the host. If genetic modificationis desired, after genetic modification and before transplant, thosegenetically modified cells may be expanded and/or selected based on theincorporation and expression of a dominate selectable marker. Transplantcan be back into the hepatic compartment or an ectopic or heterotopicsite. For transplant into the hepatic compartment, portal vein infusionor intrasplenic injection could be used. Intrasplenic injection may bethe administration route of choice because hepatic progenitorstransplanted via an intrasplenic injection move into the hepaticcompartment.

Additional medical procedures may assist in the efficacy of hepaticengraftment of the transplanted hepatic progenitors. Animal models havedemonstrated that in partial hepatectomy, administration of angiogenesisfactors, and other growth factors aide in the engraftment and viabilityof the transplanted hepatocytes. An alternative approach is totransplant the genetically modified progenitors to an ectopic site.

To date, there have been problems associated with hepatic cell therapyapproaches including sourcing of the cells, inability to cryopreservecells, emboli formation, and immunological rejection, etc.) The problemswith current hepatic cell therapy approaches may be due to the fact thatthe donor cells being used are predominantly adult liver cells and areshort-lived after isolation and reinjection. In addition, the use ofadult cells results in strong immunological rejection. The progenitorscells of the instant invention offer greater efficacy because of theirlimited capacity to elicit immunological rejection phenomena, theirability to be cryopreserved and therefore offering opportunities totissue type them (and thereby match the donor cells to the recipient)and to offer an “off-the shelf” product, and because of their extensiveregenerative potential.

With respect to gene therapy, the ongoing efforts make use of “targetedinjectable vectors,” the most popular route for clinical therapies underdevelopment. These approaches have had limited efficacy due both toimmunological problems and transient expression of the vectors. The onlyroutes for gene therapy that have proven merit-worthy have been ex vivogene therapy and have been done almost exclusively in hemopoieticprogenitor cells. We predict that ex vivo gene therapy with progenitorscells (or use of injectable vectors somehow targeted to thoseprogenitors) will prove more effective, since the vectors can beintroduced ex vivo into purified progenitor cells; the modified cellsselected and reintroduced in vivo. The advantages of the progenitorcells are their enormous expansion potential, their minimal, if any,induction of immunological reactions or ability to be tissue typed andtherefore matched to the recipient's immunological phenotype, and theirability to differentiate to produce both hepatocytes and biliary cells.

10. Other Uses

The uses for human hepatic primitive and proximal hepatic stem cells aremany and diverse. They include: 1) research on human cells; 2)production of vaccines or antivirals; 3) toxicological studies; 4) drugdevelopment; 5) protein manufacturing (using the cells as hosts forproduction of various human-specific factors); 6) liver cell therapies;7) liver gene therapies; and 8) bioartificial livers that can be used inresearch, toxicological and antimicrobial studies, proteinmanufacturing, or clinically as a liver assist system. Considering theability of the primitive and proximal hepatic stem cells todifferentiate into hepatocytes and biliary cells, the cells of thepresent invention can be used both for hepatic or bliary fates dependingupon the microenvironment in which they are placed.

The availability of human hepatic progenitor cells (all four categories)will enable much more extensive research on human cells, will facilitatethe development of successful forms of liver cell and gene therapy, andshould enable the development of human bioartificial livers for use bothin research and as clinical assist devices. At present, the limitedsupply of healthy human tissues precludes clinical programs in livercell therapy or in human bioartificial livers. The progenitor cellpopulations should have sufficient expansion potential to overcome, orat least greatly alleviate, that limited supply. Moreover, these cellsand their immediate descendents show preferential survival to ischemia,both cold and warm, relative to that observed with mature liver cells,meaning that livers that cannot be used for liver transplantation or forproducing healthy mature liver cells are sources of the progenitorcells.

The invention is illustrated by the following non-limiting examples.

Example 1 Preparation of Hepatic Cell Suspension From Fetal Tissue

Liver tissue was obtained from fetuses between 18-22 weeks gestationalage obtained by elective terminations of pregnancy. The samples of livertissue were shipped overnight in RPMI 1640 supplemented with 10% fetalbovine serum.

Tissue volume ranged from 2 to 12 mL after a preparatory wash in cellbuffer (RPMI supplemented with bovine serum albumin (BSA Fraction V,0.1%, Sigma, St. Louis, Mo.), selenious acid (300 pM), and antimicrobialmix, AAS (Gibco BRL/Invitrogen Corporation, Carlsbad, Calif.). Livertissue was subdivided as necessary into fragments of 3 mL or less fordigestion in 25 mL of cell buffer containing type IV collagenase anddeoxyribonuclease (Sigma, St Louis, Mo.; both at 6 mg per mL).Incubation was conducted at 32° C. with frequent agitation for 15-20minutes and resulted in a homogeneous suspension of cell aggregates. Thesuspension was then passed through a 40 gauge sieve and spun at 1200 RPMfor five minutes before resuspension in a calcium-free solution of Hanksbuffered salt solution, supplemented with EGTA (0.2 mM, Sigma), Hepes(20 mM, Boehringer Mannheim), BSA (0.1% Sigma), DNase (0.01% Sigma) andtermed HBSS mod.

The enzymatically digested suspension comprises hemopoietic and hepaticsubpopulations. An antigenic profile of the enzymatically digestedsuspension is shown in Table 1 with the AFP-expressing cells being 6-9%of the original cell suspension (FIG. 12) and with a comparablepercentage of albumin-expressing cells along with a significantcontamination of hemopoietic cells (see the percentages of CD45 andglycophorin A expressing cells in Table 1). If the original cellsuspension is cryopreserved, some cells, such as the erythroid cells,are lost enriching the albumin and AFP-expressing cells to 15-20% (Table1). However, the most striking enrichment occurs with the partialenzymatic digestion with collagenase to yield aggregates of parenchymalcells that are then separated from the non-parenchymal (floating) cellsby repeated low speed centrifugation as described below and yields acell suspension that is more than 80% albumin and AFP-expressing cells(Table 1).

Hematopoietic cells (mostly erythrocytes and erythroblasts) and floatingnon-parenchymal cells were then separated from the parenchymal cellfraction by repeated slow speed centrifugation at 30 g (300 RPM) forfive minutes in HBSS mod. The pellet was resuspended and re-centrifugedin 40 mls of HBSS mod until the color showed minimal contamination withred blood cells. Normally, as reported by others, this required four orfive cycles of centrifugation and resuspension [14, 15]. Clumping wasminimized by a second-round of enzymatic digestion in fresh collagenasesolution followed by sieving through a 50 μm nylon mesh and return ofthe cells to a calcium-free buffer.

The resulting cell suspension was washed twice then 5 mL aliquots, eachcontaining about 2×10⁷ cells, were layered onto 5 mL of Ficoll Hypaque(Amersham Pharmacia, Piscataway, N.J.) in 50 mL Falcon tubes and spun at3000 RPM for 20 minutes. Cells from the interface and pellet wereresuspended separately in plating media (RPMI supplemented) and analiquot of each was stained with trypan blue for enumeration andviability assessment with a hemacytometer. Cell viability was routinelyhigher than 95 percent. The low-speed centrifugation method forenrichment of the parenchymal cells eliminated the hemopoieticconstitutents leaving a cell suspension that was approximately 80%AFP-expressing cells. The majority of the AFP-expressing cell areproximal hepatic stem cells given that they express AFP, albumin andCK19 but not hemopoietic markers (Table 1).

TABLE 1 Flow Cytometric Analyses on Freshly isolated Fetal Liver Cells %Positive % Positive in Original in Enriched Cell Suspension, OCSParenchymal Marker Location (% in C-OCS) Preparation AFP Cytoplasmic 6.4± 0.8% (~15-20%) 75.5% Albumin Cytoplasmic 9.3% (~15-20%)  >80% CD45Surface 1.4% ± 0.4 Glycophorin A Surface >50% (37.5 ± 9%) NegligibleCD34 Surface (2.7 ± 0.5%) CD38 Surface (1.2 ± 0.3%) CD14 Surface 3.0± CD117 (c-kit) Surface ~1% Ep-CAM Surface n.d. CD146 Surface n.d. N-CAMSurface n.d. CAM-5.2 Surface n.d. PE-CAM Surface n.d. CD133 Surface n.d.Cytokeratin 19 Cytoplasmic n.d. Cytokeratin 8/18 Cytoplasmic n.d. OCS =original cell suspension; C-OCS = original cell suspension wascryopreserved in a proprietary buffer. The cells were later thawed andthen analyzed for expression of the markers. A number of cells,especially the erythroid cells (enucleated subpopulation) do not survivecryopreservation. Parenchymal preparation = after elimination oferythroid cells and other floating, nonparenchymal cells by repeatedcentrifugation at low speed spins; n.d. = not done

Example 2 Preparation of Hepatic Cell Suspension From Adult Tissue

A human liver was obtained from an authorized organ procurementorganization. The donor was a 13 year old female who had suffered braindeath. The liver was digested using a whole-organ perfusion technique.The single-cell suspension was then fractionated to obtain viable cellsusing a 2-step Optiprep gradient (9-12.5%) on a Cobe 2991 cell washer.Live cells were then separated from residual dead cells by mixing equalvolumes of the 9% (band 1) and 12.5% (band 2) fractionated cellsindividually with 25% Optiprep for further fractionation on the Cobe2991. Based on flow cytometric analysis of forward and side scatterparameters, the cellular composition of bandl and band 2 appearedsimilar. The cells were cryopreserved.

Example 3 Colony Formation From Adult Human Liver Cells

To assess the presence of liver stem cells by colony formation, cellsfrom Example 2 were thawed and plated at a density of 12,500 livecells/well on a 6 well plate, in triplicate, onto a STO-5 feeder layer.The tissue culture medium used was DMEM F12 containingpenicillin/streptomycin (50 U/ml/50 ug/ml), bovine serum albumin (0.2%w/v), transferrin (10 ug/ml), free-fatty acids (7.6 uEq/L) nicotinomide(4.4 mM), selenium (3×10(−8) M), copper (1×10(−6) M), 2-mercaptoethanol(5×10(−5) M), L-glutamine (2 mM), insulin (5 ug/ml), hydrocortizone(10(−7) M), with (+EGF) or without (−EGF) the addition of epidermalgrowth factor.

Cells were cultured for 5 days, fixed, and colonies counted asvisualized by light microscopy. No colonies were observed in any wellsof unfractionated cells. This may be due to inhibitory effects of deador dying cells, or some other component of the cell preparation prior tocentrifugation on Optiprep gradient. However, colonies were observedfrom both the band 1 and band 2 cells fracdtions. 8 total colonies wereobserved in the 3 wells containing cells from band 1 (4 from +EGFmedium, 4 from −EGF medium), and 13 total colonies were observed in the3 wells from band 2 (11 from +EGF medium, 2 from −EGF medium). Theoverall frequency of colony forming cells calculated for this experimentwas 0.03%.

Example 4 Co-Expression of Albumin, CD133, and Ep-CAM in a Subpopulationof Adult Human Liver Cells

Cells were isolated from donor livers essentially as described inExample 2. The presence of cells expressing the CD45 cell surfaceantigen, or Leukocyte Common Antigen, a tyrosine phosphatase expressedwidely on white blood cells (leukocytes) was assessed by fluorescenceactivated cell sorting (FACS) using an anti-CD45 monoclonal antibody.Approximately 17 percent of the cells were CD45-positive (FIG. 14A). TheCD45-positive cells were depleted by magnetic cell sorting usinganti-CD45 monoclonal antibody and super-paramagnetic MACS MicroBeads andthe autoMACS, an automated bench-top magnetic cell sorter. Both themagnetic-bead labeled antibody and the instrument were supplied byMiltenyi Biotec. CD45-positive cells could also be depleted by“panning”, fluorescence activated cell sorting, or other modes ofnegative immunoselection. After depletion, the fraction of CD45-positivecells remaining in the liver cell preparation was reduced toapproximately 1 percent (FIG. 14B). Depletion of CD45-positive cellsfacilitates the further analysis of antigens on hepatocytes and hepaticprogenitor and stem cells. It also should facilitate the isolation ofenriched populations of these cells.

a. Albumin

After depletion of CD45-positive cells, a sample of the liver cells wasanalyzed for expression of human serum albumin. Cells were fixed withparaformaldehyde, permeabilized by treatment with 0.2% Triton X-100detergent, and stained by sequential incubation with a mouse IgG₁monoclonal antibody to human albumin, and affinity-purified goatantibodies against mouse immunoglobulin G₁ (IgG₁) labeled with thefluorescent dye A647. Background staining and autofluorescence of thecells was determined by using a purified mouse myeloma protein (also anIgG₁), with no specific binding activity to human antigens, in place ofthe anti-albumin monoclonal antibody. Approximately 97.5 percent of thecells were albumin-positive (FIG. 15A). The gating for positive staining(red outline) was determined by comparison to the mouse myeloma proteincontrol (not shown).

Measurement by FACS of forward light scatter and side light scatter canbe used to characterize cellular populations. The forward and sidescatter are primarily functions of cellular size and intracellularstructural complexity, respectively. As shown in FIG. 15B, thealbumin-positive cellular population from adult human liver comprises amajority class of cells with relatively high forward (FSC) and sidescatter (SSC). Analysis of size and morphology, as well as additionalbiochemical and antigenic markers (not shown), shows that these cellshave properties consistent with mature, small hepatocytes (average sizeapproximately 18-22 micrometer diameter). The largest hepatocytes(approximately >30 micrometer diameter) from normal adult human liverapparently are under-represented in our preparations, probably becauseof greater susceptibility to death during the period between harvestingof the organ and perfusion, and/or greater susceptibility to damageduring the isolation procedure. However, FIG. 15B also shows that, inaddition to the mature, small hepatocytes, many cells characterized bylower forward and side scatter also express albumin. The few(approximately 2.5 percent) albumin-negative cells in the preparationalmost exclusively show very low forward and side scatter (FIG. 15C).These may be dead cells, or very small cells such as late stageprecursors of red blood cells.

b. CD133

The antigen CD133 (AC133) is a cell surface glycoprotein of 120kilodaltons that has five transmembrane domains. The protein is similaror orthologous to the mouse protein prominin. The human CD133 antigenoriginally was identified on a subset of early progenitors, includingstem cells, in the lineage of blood-forming (hematopoietic) cells.Certain other immature cells express CD133, including developingepithelium in human embryos (week 5), endothelial cell precursors, andneuronal progenitors or stem cells. Expression of CD133 also has beenreported on certain human tumors and tumor-derived cells lines, such asretinoblastomas and the colon carcinoma line CaCo-2. The protein isfound concentrated preferentially in plasma membrane profusions such asmicrovilli. When found on epithelial cells, it localizes preferentiallyto the apical, but not to the baso-lateral membrane surface. Previousstudies, in particular by immunohistochemistry, have failed todemonstrate CD133 protein expression in adult human epithelial tissue,despite the presence of detectable messenger RNA for the protein in manyhuman tissues, including adult liver.

We used staining with a fluorescent-labeled monoclonal antibody andanalysis by FACS to search for cells expressing the CD133 antigen in ourCD45-depleted adult human liver cell preparations. Surprisingly, inlight of the prior negative reports, we observed that a majority of theCD45-depleted liver cells (see FIG. 14B) show positive staining forCD133. FIG. 16A reveals approximately 58 percent CD133-positive cells ina preparation from the liver of a juvenile (two year-old) individual.The presence of a substantial population of CD133-positive cells,including cells of the size of small mature hepatocytes, has beenobserved in cell preparations from additional individuals, includingadults. The CD133-positive population (upper box in FIG. 16A) comprisesroughly half of the cells in the preparation identified as mature(small) hepatocytes on the basis of side light scatter (FIG. 16A) andforward light scatter (not shown). It also comprises many cells that aresmaller than and morphologically distinct from mature hepatocytes, asjudged by light scatter.

Magnetic cell sorting can be used to positively select the liver cellsthat express CD133. FIG. 16B shows enrichment of CD133-positive cells toapproximately 75% of recovered cells after one cycle of magnetic sortingutilizing the autoMACS instrument (Miltenyi Biotec). The use of higheramounts of antibody-coupled MACS MicroBeads and adjustment of thesorting conditions should permit the isolation of more highly enrichedCD133-positive cell populations with nearly quantitative yield. {Alsonote that other methods of positive immunoselection can be used toenrich for the CD133-positive cells}. As judged by side scatter (FIG.16B and forward scatter (not shown), the enriched CD133-positive cellscomprise all of the CD133 subpopulations identified in the CD45-depletedliver cell preparation.

c. Ep-CAM

The epithelial cell adhesion molecule (Ep-CAM, also known as GA733-2,CO17-1A, EGP40, KS1-4, and KSA) is a glycoprotein implicated inhomophilic, calcium ion-independent cell-cell adhesion. The protein isexpressed in many human epithelial tissues, and appears to beup-regulated substantially in proliferating epithelial cells, includingtumor cells. C. J. de Boer and colleagues reported that in 8-weekembryonic human liver most hepatocytes express detectable Ep-CAM protein[de Boer CJ, van Krieken J H, Janssen-van Rhijn C M, Litvinov S V.(1999). “Expression of Ep-CAM in normal, regenerating, metaplastic, andneoplastic liver,” Journal of Pathology 188:201-6]. By contrast, innormal adult human liver they failed to detect Ep-CAM expression inhepatocytes, and reported that only bile duct epithelial cells stainpositively for this antigen. Finally, the antigen was detected in cellsidentified as hepatic precursors in situations in which liverregeneration and repair was induced by biliary cirrhosis, and in cellsof certain liver tumors, particularly cholangiocarcinomas.

By FACS analysis we consistently detect a minor population ofEp-CAM-positive cells in unfractionated human liver cell preparationsfrom both juveniles and adults. The Ep-CAM-positive population comprisesapproximately 0.4 to 2.5 percent of the cells. As shown in FIG. 17Ep-CAM-positive cells also can be observed in liver cell populationsafter depletion of >95 percent of CD45-positive cells. FIG. 17B showsthe Ep-CAM-positive cells from one such human liver preparation (0.57percent in the region of the plot gated as shown by the red outline, ascompared to 0.15 percent in the same gated region for a control antibodythat does not stain any known human antigen, FIG. 17A; thusapproximately 0.57-0.15=0.42 percent of the cells are Ep-CAM-positive).Double label analysis (data not shown) demonstrates that the vastmajority of the Ep-CAM-positive cells in the CD45-depleted populationare, as expected, CD45-negative. However, it appears that some (roughly1 percent) of the CD45-positive cells in our human liver preparationsalso express Ep-CAM (data not shown).

d. Co-Expression of Ep-CAM, CD133, and Albumin

We searched for liver cells from adult human liver that express bothEp-CAM and CD133. Cells were incubated with monoclonal antibodies toCD133 and Ep-CAM, each directly conjugated with a differentfluorochrome. As shown in FIG. 17C, approximately 42 percent of thecells in this particular CD45-depleted adult human liver cellpreparation stained detectably for CD133. (The somewhat lower degree ofCD133 staining here than in the experiment shown in FIG. 16A may resultfrom actual differences between liver cell preparations from differentdonors, as a consequence of age or other variables, or from unidentifiedvariations in experimental technique). Among cells in the populationthat stained strongly for Ep-CAM (shown within the red boundary in FIG.17B), approximately 70 percent also stained positively for CD133 (FIG.17D). Thus, in this particular liver preparation approximately 0.3percent of the total CD45-negative cells co-expressed Ep-CAM and CD133.

The cell preparation used for the experiment shown in FIG. 17 wasidentical to that used in the analysis of albumin expression shown inFIGS. 14 and 15. As noted above, approximately 97.5 percent of the cellsin the CD45-depleted cell population stained positively for albumin, andthe few albumin-negative cells displayed a distinctive pattern of lowforward scatter and side scatter. As shown in FIG. 17E, virtually all(approximately 99.5 percent) of the cells found to co-express CD133 andEp-CAM showed forward scatter and side light scatter characteristic ofthe albumin-positive cells; they fall entirely outside of the boundedregion of the plot of forward scatter versus side scatter that iscontained all of the albumin-negative cells (see FIG. 15C). Thus, thepostnatal human liver cells that co-express Ep-CAM and CD133 alsoexpress human serum albumin.

e. Co-Enrichment Of CD133 and Ep-CAM Expressing Cells

As shown in FIG. 16B, positive immunoselection such as magnetic cellsorting allows the enrichment of CD133-positive cells from human livercell preparations. We assessed cells in the starting population (alreadyCD45-depleted) and the CD133-enriched preparation for the expression ofEp-CAM. FIG. 17A shows that at least 1.1 percent (deliberated gatedtightly) of the starting population expressed Ep-CAM. After enrichmentof CD133-positive cells, the resulting population (FIG. 5B) contains atleast 4.5 percent Ep-CAM-positive cells. This confirms the coexpressionof CD133 and Ep-CAM in a subpopulation of cells from adult human liver,and demonstrates that these cells can be enriched by positiveimmunoselection. Analysis of forward and side scatter (as in theexperiment of FIG. 17) by the cells that co-express the two surfaceantigens again shows that nearly 100 percent of these cells also must bealbumin-positive.

The adult human liver cells described above co-express albumin, aprototypical marker of the hepatocyte lineage, together with eitherCD133, or Ep-CAM, and therefore have the same is the same phenotypicprofile of certain hepatic stem cells from human fetal liver describedherein. Moreover, the adult human liver cells described herein are livercells of size smaller than mature hepatocytes (even “small hepatocytes”of 18-22 micron diameter). Taken together with the finding that adulthuman liver contains cells capable of forming colonies under conditionsthat operationally define hepatic stem cells (i.e., in serum-free mediumwith STO feeder cells), the co-expression of albumin, Ep-CAM and CD133demonstrates the presence of such stem cells in adult liver. Methods ofpositive immunoselection described herein may be used to isolate cellsthat simultaneously express the two surface markers, Ep-CAM and CD133 inorder to obtain highly enriched populations of hepatic stem cells fromhuman liver, including tissue derived from a child or an adult.

Example 5 Primary Cultures of Proximal Hepatic Stem Cells on STO FeederLayers

Most of the liver progenitors, with the exception of the primitivehepatic stem cells, do not survive for long being co-cultured withembryonic liver stromal feeders; feeders from neonatal livers, adultlivers, or diverse adult tissues were not successful (Sigal et al, 1994;Brill et al, 1995; Sigal et al, 1995; (Brill S, Zvibel I, and Reid L M.Expansion conditions for early hepatic progenitor cells from embryonaland neonatal rat livers. Digestive Diseases and Sciences 44:364-371,1999). The embryonic liver stromal feeders can be replaced by STO cells,an embryonic stromal cell line, used as routine feeders for embryonicstem cells and found to support clonogenic expansion of freshlyisolated, normal rodent hepatic stem cells and diploid adult rat livercells (Kubota and Reid, 2000). These conditions were found essentialalso for the all of the progenitors from human fetal liver, with theexception of the primitive hepatic stem cell that would expand with andwithout the feeders (Moss et al submitted). The STO feeders have alsoproven successful for hepatic progenitors from neonatal and adult humanlivers (Ludlow, et al, in preparation). The factors supplied by theembryonic stromal feeders and essential for the progenitors are notknown.

STO Feeders originally from ATCC were expanded from stock cells in 75 cmflasks in DMEM/F12 (Gibco/BRL/InVitrogen Corporation, Carlsbad, Calif.)supplemented with 10% fetal bovine serum, FBS (Hyclone, Logan, Utah) and1% DMSO (Sigma, St. Louis, Mo.). After three passages to provide nineconfluent flasks, the cells were treated for 2 hours with 10 μg/mLmitomycin C (Sigma, St. Louis, Mo.; also, Biomol, Plymouth Meeting, Pa.)to induce cell cycle arrest and washed twice with culture medium. Thecells were trypsinized and resuspended in cryopreservation medium (50%DMEM/F12, 40% FBS, 10% DMSO) before freezing in 1 mL aliquots of 5×10⁶cells and stored at −80° C. Feeders were prepared by seeding 6×10⁴thawed cells/cm² onto culture plates pre-coated with 0.1% gelatin(Sigma, St. Louis, Mo.). Detailed protocols described in [16] areincorporated herein by reference.

Cells passaged onto STO cells were cultured in a serum-free, hormonallydefined medium (HDM) comprising RPMI 1640 (GIBCO/BRL/InVitrogenCorporation, Carlsbad, Calif.) supplemented with 0.2% bovine serumalbumin (Fraction V Fatty acid free, Sigma, St. Louis), insulin (5μg/ml), transferrin/Fe (10 μg/ml), selenium (3×10⁻⁸M), 2 mercaptoethanol(5×10⁻⁵ M) a complex mix of free fatty acids (7.6 μEq; [16, 17]),hydrocortisone (10⁻⁷ M), glutamine (2 mM), nicotinamide (4 mM), and AAS(penicillin, 1000 μg/mL, streptomycin 100 μg/ml, and amphotericin B 250ng/mL, Sigma). Preferably, neither cytokines classic hepatic growthfactors (e.g. epidermal growth factor, EGF, hepatocyte growth factor,HGF, nor insulin-like growth factors, IGFI and IGFII) were used.

Primary cultures of dispersed, enriched parenchymal cells from Example 1were plated onto a STO feeder layer produced stable aggegates ofproximal hepatic stem cells that express albumin, AFB and CK19. Typicalcells staining for albumin, AFP, and CK19 are shown in FIGS. 6 a-6 c.These cells were also positive for CK8/18. Unlike cells cultured on aplastic substratum as described in Example 6, the proximal hepatic stemcells seeded onto STO feeders retained a consistent morphology andmaintained AFP expression for several weeks. Since these conditionssupport both proximal hepatic stem cells and more differentiated cellsincluding diploid adult liver cells ([7]) co-culture with STO feedersproved unsuitable for selection of truly primitive colony-forming cells.

Example 6 Selection of Primitive Hepatic Stem Cells

The enriched parenchymal cell suspension of Example 1 was plated at adensity of 2000-5000 cells/cm² onto tissue culture plastic in aserum-free medium supplemented with lipids, insulin and transferrin/Fe(HDM). For the first 12 hours after plating, the medium contained 10%FBS to promote cell attachment after which the cultures were maintainedserum-free. Media changes occurred at three-day intervals.

Immediately after attachment the predominant cells present in culturewere proximal hepatic stem cells and committed progenitors, aggregatesof cells with a classic parenchymal cell morphology, and with expressionof albumin, AFP and/or CK19; the proximal hepatic stem cells willdemonstrate albumin, AFP and CK19 (FIG. 2 a). After several days, theproximal hepatic stem cells and committed progenitors ceased expressingAFP and were replaced by solitary, motile cell types with amyofibroblastic appearance that dispersed into the dish. In addition toproximal hepatic stem cells, several other cell types were present inculture, some solitary, some forming extensive confluent monolayers,while others formed discrete round cell groupings. Amongst these typesof cells, positive staining for albumin was observed only in theproximal hepatic stem cells, the committed progenitors, and in circular,tightly aggregated colonies, the primitive hepatic stem cells, whichappeared in culture concurrently with the gradual demise of the proximalhepatic stem cells and the committed progenitors.

Colony formation showed a predictable sequence of events. An initialwave of colonies appeared within the first few days in culture andappeared to arise from aggregations of pre-existing cells (FIG. 1 b).However, after 5-7 days, a new wave of colony formation started fromsolitary cells scattered throughout the culture dish. These colonieswere first recognizable as groups of 4-8 small, dark, tightly compactedcells with lamelipodia at the periphery that formed a narrow continuousfringe (FIG. 2 a). The colonies expanded into extensive groupings oftightly aggregated, rounded cells 8-10 um in diameter (FIG. 2 b-2 e).The general appearance of these late-forming colonies is distinct fromcolonies that form in the first days of culture, which were composed oflarger cells, and from the initial aggregations of proximal hepatic stemcells that constitute the main parenchymal cells in fetal liver.

The primitive hepatic stem cells grew well on tissue culture plastic inthe HDM and achieved diameters of up to 1 cm after several weeks inculture. Numerous colonies were removed selectively and dispersed bytrypsinization to yield an average cell number per colony that rangedfrom 1000 cells for colonies 3 mm in diameter to 15,000-20,000 in largecolonies with diameters of 1 cm.

Typically, the outermost cells of the colonies converted into aflattened phenotype that became separated from the colony to formsolitary, large diameter cells that dispersed throughout the culturedish (FIG. 3 a). In other colonies, cells at the perimeter assumed anelongated, fibroblastic appearance that initially wrapped closely aroundthe circumference of the colony, perhaps tightly associated mesenchymalcells (FIG. 3 b). These cells also migrated away from the colonies asisolated, fusiform cells. These dispersed cells remained highlyproliferative and often became the predominant cell type in culture,forming a tightly packed layer that extended throughout the dish. Thecolonies were surrounded but not overgrown by this cell layer, though atransitional zone formed at the margin of each colony where the two celltypes became interspersed (FIG. 3 c).

Example 7 Antigenic Profiles of Colony Forming Cells in Plastic Culture

The antigenic characteristics of the cells cultured in Example 6 wasinvestigated with immunocytochemical staining for markers relevant tohepatic organogenesis. Cell cultures were fixed with a 50/50 (V/V)mixture of methanol and acetone for 2 min at room temperature. Severalstaining regions were created on the surface of each dish with a PAPmarker pen (Research Products International Corp, Mt. Prospect, Ill.) toallow multiple antibody combinations within the same culture.Non-specific binding sites were blocked by incubation with a solution of10% goat serum (GIBCO/BRL/In Vitrogen, Carlsbad, Calif.) in PBS for 30min at room temperature. After rinsing twice with PBS, primarymonoclonal antibodies were applied to each of the staining regions(normally 0.1-0.3 mL per region) and incubated overnight. Afterincubation overnight at 4° C. cells were washed twice with PBS and thenincubated with a secondary antibody conjugated either to Alexa 488(1:750) or Alexa 594 (1:1250) (Molecular Probes, Eugene Oreg.). In someinstances a primary monoclonal antibody conjugated to either FITC or PEwas available and provided the means for double labeling by incubationwith this antibody after completion of the labeling protocol with anunconjugated primary antibody.

The antigenic profile is summarized in Table 2. Colonies stainedpositive for a number of markers previously linked to hepatic cell typesincluding albumin (FIG. 4 a), CK19 (FIG. 4 b), epCAM (FIG. 4 c) NCAM(CD56, FIG. 4 d) but not PECAM (CD31; data not shown.). c-kit stainingwas seen in several colonies, generally localized to a narrow segment atthe margin of colonies (FIG. 5 a). Also, colonies were positive for theputative stem cell marker, CD133 (AC133, FIG. 5 c). Interestingly, thecells in the transitional zone at the periphery of colonies stainedpositive for a recently described endothelial marker, CD146 (M-CAM, FIG.5 c), and remained positive for this protein while in the vicinity ofthe colonies, possibly identifying a closely associated mesenchymal celltype, possibly an endothelial progenitor. The primitive hepatic stemcells that emerged as colonies were negative for AFP, indicating thatthe primitive hepatic stem cells are a precursor to proximal hepaticstem cells, which in turn are precursors to hepatocyte progenitors andbiliary progenitors.

TABLE 2 Phenotypes of Cultured Cells Ductal Plate Proximal hepaticBiliary Phenotype Stem Cells stem cells Hepatocytes Epithelia MorphologyDark, tightly Transiently are Do not survive the stringent understringent packed cells, 7-10 aggregates of conditions conditions: indiameter, with small, cuboidal tissue culture “pincushion” cells thatbecome plastic and morphology motile HDM Morphology on Dark, tightlyStable, densely Diploid subpopulation flatten STO feeders packed cells,7-10 packed and have distinct cellular and in HDM in diameter, withaggregates of boundaries; polyploid cells “pincushion” cells survive butdo not grow morphology Alpha- Not Expressed +++ Not expressed Notexpressed fetoprotein Albumin +++ +++ +++ Not Expressed CK8/18 +++ ++++++ +++ CK19 +++ +++ Not expressed +++ c-Kit +++ (cells at the Notexpressed Not expressed Not expressed periphery of the colony) Ep-CAM+++ +++ N-CAM +++ (cells at the Not expressed Not expressed Notexpressed periphery of the colonies) CAM-5.2 +++ (some, not Notexpressed all, of the cells CD133 +++ +++ Not expressed Not expressed(AC133) CD146 Periphery of Not expressed Not expressed Not expressedcolonies (unknown if this an associated mesenchymal cell or derived fromthe primitive hepatic stem cell) PE-CAM Not expressed Not expressed Notexpressed Not expressed

Example 8 Passage of Colony Cells From Plastic Substratum to STO FeederCells

STO feeders were used to assess the fates of the primitive hepatic stemcells after selective passage from the plastic substratum. After 1-2weeks in culture, colonies on plastic substratum from Example 6 werephysically lifted from plastic substrata by aspiration into a 100 uLpipette under binocular magnification. Up to 50 colonies were collectedin HBSS mod and then digested for up to 20 min in collagenase solutionwith agitation to disperse cells into suspension.

Colony forming efficiency following passage from plastic to a STO feederlayer was low, ranging between 0.5 and 1% for cells passaged atdensities of 500 or 50 cells per cm². Initial attachment of passagedcolony-forming cells was improved by the presence of EGF (20 ng/mL) inthe plating medium. The low colony forming efficiency may have been due,in part, to the need to subject the cells to lengthy (up to 20 minutes)collagenase digestion in order to achieve single cell suspensions.

After passage onto STO feeders cells, the primitive progenitor cellsmerged into the STO cell layer and reappeared as tightly compactedcolonies of small cells after 4-5 days in culture (FIG. 6 a). The newcolonies enlarged over the following weeks to produce tightly aggregatedcircular groupings, often with a slight thickening at the circumference(FIG. 6 c). In some colonies a secondary proliferative stage occurred inwhich an eruption of cells occurred from a point at the edge of thecolony and spread out over the STO layer, often surrounding the originalcolony (FIG. 6 c).

The immuno-cytochemical properties of colonies formed on STO cells werethe same as those described above for colony-forming cells on plasticsubstrata. This includes positive staining for albumin, CK19, CK18, andCD133. As in initial colonies raised on plastic, markers such as CD146and NCAM were most clearly expressed at the periphery of the coloniesformed on STO cells. This marginal expression pattern became even morepronounced when colonies were surrounded by cells that proliferated fromthe primary colony. This is shown clearly for NCAM in FIG. 9 where theinterface between the original colony formed from passaged cells and thesecondary proliferation is marked by a band of cells with intenselypositive NCAM expression. This pattern was also seen for expression ofthe pan cytokeratin marker CAM 5.2 and double labeling for NCAM and CAM5.2 showed that the two markers were expressed at high levels in thesame region of marginal cells (FIG. 9).

Finally, the characteristics of the erupting cell type was of interestas they emerged from the colonies in a distinct arrangement consistingof a parallel row of cells that enlarged into a branching pattern oflinearly arranged cells with clear intercellular spaces (FIG. 8). Whenthese cells had enlarged into an extensive sheet around the originalcell colonies they appeared to lose the linear organization apparent atthe time of emergence. However, staining for albumin revealed that theorganization into rows of cells was maintained within the cell mass(FIG. 10 a), and co-staining for CD146 showed that this marker is alsoexpressed at high levels within the proliferating cell group (FIG. 10b). Perhaps of most significance in these cells is the appearance ofstaining for AFP at the periphery of the emerging cells (FIG. 11. Thisrepresents the first point in the ex vivo manipulations described hereinthat AFP expression can be linked to the progeny of the initialcolony-forming cells. These data indicate that the primitive hepaticstem cells isolated in Example 6 may be used to produce hepaticcommitted progentors.

Example 9 Presence of Primitive and Proximal Hepatic Stem Cells in aLiver from a Neonatal Donor

A liver was obtained from a donor who was born after approximately 28weeks of gestation and survived only one day. The period of warmischemia between cardiac arrest and the harvesting and flushing of thedonor organ was between six and seven hours. Thereafter the organ wasmaintained on ice for approximately twelve hours. The whole organ (wetweight approximately 100 grams) was subjected to perfusion and digestionwith Liberase, and a cell suspension was prepared, essentially as for aliver obtained from a human child or adult. Non-viable cells and manyred blood cells were removed by preparative centrifugation usingOptiprep. However, it was difficult to determine the actual yield andpercentage of viable, non-erythroid cells in the final preparation.

Portions of the recovered cells were depleted further of red blood cellsby differential centrifugation, essentially as described for fetal livercells, and then seeded in culture under conditions appropriate todetermine the presence of primitive hepatic stem cells (i.e., plating inserum-free, hormonally defined medium on a tissue culture plasticsubstratum). Other portions of cells were plated, without furtherpurification, under conditions to assay for proximal hepatic stem cells(i.e., plating in serum-free defined medium with STO feeder cells).Additional portions of cells were seeded on tissue culture plasticcoated with Type I collagen, in serum-free, defined medium containing orlacking supplementary epidermal growth factor (EGF) at 0.5 ng/ml.

After appropriate periods of incubation, the growth of hepatic colonieswas observed in all conditions tested. In the respective assays forprimitive and proximal hepatic stem cells, the colony morphology andrate of growth was similar to that observed for cells cultured fromhuman fetal liver (generally obtained after <22 weeks of gestation)cultured under the same conditions. Representative colonies were testedby immunofluorescence staining for the expression of human albumin, andwere all positive for this marker.

Colonies of apparent epithelial (presumptively hepatic) morphologyappeared on collagen-coated plates in the presence of EGF. Additionalcells of less well-defined morphology also grew rapidly in thesecultures, but have not yet been characterized in detail.

Colonies of presumptive epithelial cells also appeared oncollagen-coated plates in the absence of supplementary EGF. Some ofthese colonies were picked individually, using a manual pipettingdevice, and transferred into fresh medium in 96 well plates. Cells fromcertain colonies have continued to proliferate in such cultures, and arebeing passaged as cell strains. It appears likely that these representstrains, potentially clonal, of propagable hepatic precursors, perhapsstem cells. Further characterization of expression of antigens includingCD133, Ep-CAM, albumin, AFP, and CK19 will determine the relative stateof differentiation of these putative hepatic stem cell lines.

Example 10 Flow Cytometry Sorting and Flow Cytometric Analyses(FACscans)

FACscans, of cytoplasmic antigens (e.g. albumin, AFP) were done withcells fixed and permeabilized with 3% paraformaldehyde prior to stainingwith the antisera. Cells were stained as indicated forimmunofluorescence but using antibodies directly labeled with therelevant fluoroprobe (see Tables 3 and 4). The flow cytometry wasperformed on a Cytomation “MoFlow” flow cytometer (Fort Collins, Colo.)(FACs facility directed by Dr. Larry Arnold). The sheath fluid wasunmodified HBSS. The MoFlow cytometer is capable of analysis or ofsorting 40,000 cells/second, with up to 12 parameters in parallel (6“colors” in combination with forward scatter and/or side scatter) andwith an accuracy of greater than 99%. For most sorts a 4W argon laserwas used with 60 mW of power and with a 100 um nozzle. Fluorescentemissions at 488 nm excitation were collected after passage through a530/30 nm band pass filter for FITC. Fluorescence was measured bylogarithmic amplification. Cells were considered positive whenfluorescence was greater than 95% of the negative control cells. Adetector value of E−1 was used for forward scatter (FSC) with amid-range amplification and, and the detector was used mid-range forside scatter (SSC) with an amplification of 1. The SSC gatings were doneby means of linear amplification with division of parameters into 256arbitrary units. Unstained cells, cells stained with an irrelevantantibody and the same fluoroprobe or with the same antibody but with nofluoroprobe were used as negative controls. In each sample,30,000-50,000 cells were assayed. Positive cells, those with greaterfluorescence than the negative controls, were evaluated further forgranularity, size, and extent of fluorescence. Cells before and aftersorting were maintained at 4° C. in the HDM to which 10% serum wasadded.

TABLE 3 Monoclonal Antibodies Antibodies (all prepared in Isotype/Target antigen mice) Dilution (all human) Commercial source CD45 IgG₁Kappa/ Common leucocyte antigen on Pharmingen (31254X; 31255X) 1: allhemopoietic cells CD 235A IgG_(2b) Kappa/ Glycophorin A (red blood cell(32591A) 1: antigen) CD14 (APC) IgG_(2a) Kappa/ Antigen present onmonocytes, 1: dendrites, (one of the endotoxin receptors) CD34 (34374X)IgG₁ Kappa/ Stem cell antigen present on 1: diverse progenitorpopulations CD38 IgG₁ Kappa/ Antigen present on B cells, (31015X;31014X) 1: thymocytes and activated T cells CAM 5.2 IgG_(2a)/1:500 CAMon ductal plate CD117 (CD11704) IgG₁/1: c-kit: receptor for stem cellCaltag (MHCK04) factor CD31 IgG₁/1:250 PE-CAM: CAM on endothelia ALB(A-6684) IgG_(2a)/1:120 Albumin Sigma, St. Louis, CD56 IgG₁/1:250 N-CAM:CAM on certain Mo. neurons and on ductal plate AFP (18-0003) IgG₁Kappa/1:250 AFB Zymed CK 8/18 IgG₁/1:1000 Cytokeratins generic forepithelia CD146 IgG₁/1:250 M-CAM: found on endothelia Chemicon, CD133IgG₁/1: AC133, stem cell marker Mylteni Biotek, Ep-CAM IgG₁/1:750 A CAMon most epithelial Neomarkers progenitors CK-19 IgG_(2b)/1:300Cytokeratin-19, keratin specific NovCastra for biliary epithelia

TABLE 4 Fluoroprobes Absorbance Maximum/ Fluroprobes Color EmissionMaximum 11. Source FITC Green 494/525 Sigma, St. Louis, Mo Phycoerythrin(PE) Yellow 480/578 Molecular Probes, Alexa 488 Green 495/519 Eugene,Oregon 7-AAD (A-1310) Red 488/650 used without an antibody forelimination of dead cells Cy-5 Far Red 649/670 Jackson Labs, West AMCABlue 350/450 Row, Pennsylvania

For flow cytometric analysis of cytoplasmic antigens (e.g. albumin,AFP), the cells were fixed and permeablized with 3% paraformaldehydeprior to staining with the antisera. Cells were stained as indicatedabove for immunofluorescence. For analysis using 2 markers, a secondantibody labeled with a distinct fluoroprobe and not overlapping inwavelength will be used. The analysis was performed using a BectonDickenson FACscan. Cells stained only with the secondary antibody wereused as negative controls. In each sample, 30,000-50,000 cells wereassayed. Positive cells, those with greater fluorescence than thenegative controls, were evaluated further for granularity, size, andextent of fluorescence.

Example 11 Determination of Albumin and Alpha Fetoprotein Expression inHepatic Cell Suspension Obtained From Fetal Tissue

In this experiment, the expression of albumin and alpha fetoprotein inproximal hepatic stem cells and in primitive hepatic stem cells wasexamined. It was initially determined that the pellet fraction isenriched for hepatoblasts (proximal hepatic stem cells) while theinterface is enriched for colony forming cells (primitive hepatic stemcells).

Albumin expression is comparable between interface and pellet cells,both in freshly isolated cells and after 10 days of culture. However,the expression is diminished in culture. This could be due to lowerexpression or proliferation of albumin negative, non parfenchymal cells.Observations on the cultures indicates that both contribute to thispattern (FIG. 20).

AFP is strongly expressed in the freshly isolated pellet fraction andweakly expressed in the interface cells. This is consistent with theobservation that AFP is not expressed in colony cells. After 10 days inculture AFP is not detectable in pellet or interface cells. Colony cellsin culture express albumin but not AFP. This could have been due to theconditions (plastic culture) which leads to suppression of AFPexpression in all cells. However, the low AFP expression in interfacecells suggests that AFP is never strongly expressed in these cells (FIG.20). Also, when colony cells are cultured in conditions that supportprolonged AFP expression in pellet cells (STO co-culture) they are stillnegative for AFP expression. There was no crossreactivity betweenantibody binding to albumin or alpha fetoprotein, and no signal wasobserved in the empty lanes.

The results of these experiments therefore demonstrate that AFP isstrongly expressed in the freshly isolated pellet fraction and onlyweakly expressed in the interface cells. This is consistent with theobservation that AFP is not expressed in colony cells.

What is claimed is:
 1. A method of isolating a human primitive hepaticstem cell, the method comprising: (a) providing a single cell suspensionof cells derived from human liver tissue; (b) separating the parenchymalcells hematopoietic cells and non-parenchymal in the single cellsuspension; (c) culturing the single cell suspension of (b) inserum-free media comprising insulin, transferrin and lipids to allow thesingle cell suspension of cells to form colonies; and (d) selectingthose colonies which are negative for alpha-fetoprotein (AFP)expression.
 2. The method of claim 1 in which the human liver tissue isfetal tissue.
 3. The method of claim 1, in which the culturing is ontissue culture plastic.
 4. The method of claim 1, wherein the serum-freemedia further comprises hydrocortisone.
 5. The method of claim 1,wherein the serum-free media further comprises an antioxidant.
 6. Themethod of claim 5, wherein the antioxidant is selenium.
 7. The method ofclaim 1 further comprising (e) selecting those colonies positive forEp-CAM expression.
 8. The method of claim 1 further comprising (e)selecting those colonies of cells which express Ep-CAM, albumin, N-CAM,cytokeratin 19, or any combination thereof.
 9. The method of claim 1further comprising the step of removing dead cells from the single cellsuspension of cells derived from human liver tissue.
 10. A method ofisolating human primitive hepatic stem cells, the method comprising: (a)providing a single cell suspension of cells derived from human fetalliver tissue; and, in any order, (b) selecting from the single cellsuspension cells those cells that do not express alpha-fetoprotein(AFP), express Ep-CAM and express at least one of N-CAM, c-kit, CK19.11. The method of claim 10 further comprising (c) selecting those cellsfrom the single cell suspension expressing CD
 133. 12. The method ofclaim 10 further comprising (c) selecting those cells from the singlecell suspension those cells that express albumin, cytokeratin 19, orboth.